The present invention relates to the solubilization, purification and refolding of proteins, especially fusion proteins, produced by genetically-transformed bacteria and other host organisms. In the following description, the expression product of such a transformed host is called a "heterologous protein," while a "fusion protein" is the product coded for by a DNA comprised of multiple peptide-encoding sequences that are translated as a single protein.
Proteins are known to be characterized by primary, secondary, tertiary and, sometimes, quaternary structures. The primary structure of a protein is made up of about 20 kinds of amino acids, and more than 200 different primary structures have so far been established. The so-called "higher order," secondary and tertiary structures of a protein are formed, for example, by hydrogen bonding, S-S bonding or hydrophobic bonding, between amino acids which make up the primary protein structure of that protein. More than 100 kinds of tertiary structures have been established as a result of X-ray diffraction analysis of protein crystal structure.
By means of various gene-manipulation techniques, it is possible to introduce DNA into a host organism in such a way that the host produces a foreign (heterologous) protein coded for by the introduced DNA. The heterologous protein thus produced often assumes a higher-order structure which inhibits physiological activity and causes the protein to remain, in the host cells, in the form of insoluble agglomerates. Effective utilization of such insoluble protein typically requires a series of treatments, including solubilization, purification and refolding.
There are several conventional treatments for solubilizing and refolding of insoluble protein produced in genetically-transformed host cells. For example, the insoluble protein may be solubilized in a solution containing a strong protein denaturant, such as a 4 to 9 M solution of guanidine hydrochloride, and then refolded by reducing the concentration of the protein denaturant. But this approach presents the problem of how to handle a large volume of solutions, because dialysis or dilution of the protein/denaturant solution is required to lower the concentration of the protein denaturant. When a compound like guanidine hydrochloride is used, an additional problem arises in handling the resulting waste liquid, since disposal of guanidine hydrochloride cannot be effected by any usual method for waste-liquid disposal, such as the active sludge method.
According to another process to accomplish refolding, insoluble heterologous protein is solubilized in an alkali solution and thereafter refolded by decreasing the pH of the solution. This process is simpler and easier to implement than the aforementioned approach employing a denaturant, and is more readily used in handling a large amount of materials, since the lowering of the pH can be achieved by adding an acidic substance.
One problem with the acidification method, however, is slow rate of solubilization of heterologous protein, due to the lack of contact of the alkali solution with the interior of the protein agglomerates. A slow rate of solubilization is undesirable because it causes a lower yield.
Also by virtue of recent advances in genemanipulation techniques, it is known that even those proteins, which are difficult to obtain via heterologous expression in transformed host cells, can be obtained as a component of a fusion protein which further comprises another protein segment more easily expressed in the host. According to this approach, two or more polypeptides are separated, as components of a fusion product, by an amino-acid sequence that is specifically digested by a proteolytic enzyme, such as thrombin, factor Xa or trypsin, so that the desired protein can be obtained when the fusion product is later digested by the enzyme.
For treatments such as solubilization, purification and refolding, there are usually optimum conditions for each heterologous protein, including a fusion protein, that result in the desired structure. For example, an insoluble fusion protein, as can be produced by suitably transformed Escherichia coli, that is comprised of the .beta.-subunit of human nerve growth factor ("NGF") and at least a part, particularly an N-terminal portion, of the human growth hormone ("hGH") molecule, may be solubilized upon treatment with an alkali solution, but may precipitate when pH is lowered to effect refolding, occasioning a substantial decline in yield.